Lipocalins represent a family of functionally diverse, small proteins comprising 160-180 residues that share high conservation at the tertiary structural level while having weak amino acid sequence homology (Flower et al., 2000; Skerra, 2000). Their fold is dominated by an eight-stranded antiparallel β-barrel with an α-helix attached to its side, whereby four structurally variable loops, which connect neighboring β-strands at the open end of the barrel, form the entrance to a ligand pocket. Further hallmarks are three structurally conserved regions (SCRs) (Flower, 1996), which assist the identification of new lipocalins at the primary structure level.
Lipocalins were initially described for eukaryotes and only more recently identified in Gram-negative bacteria (Flower, 1996; Bishop, 2000). The bacterial lipocalin (Blc) was first discovered in Escherichia coli (Bishop et al., 1995), but sequence analyses have indicated the existence of at least 20 other bacterial lipocalins, for example in Citrobacter freundii,Vibrio cholerae and many other Enterobacteriaceae.
Blc belongs to the class I outer membrane lipoproteins, carrying a type II signal peptide at the N-terminus, which directs export into the periplasm. After signal peptide processing, the protein becomes anchored into the inner leaflet of the outer membrane (Bishop et al., 1995) via a lipid-modified amino-terminal cysteine residue. The blc promoter is mainly induced at the onset of the stationary growth phase via the rpoS sigma factor, which generally directs gene expression for adaptation to starvation and high osmolarity or other conditions known to exert stress on the cell envelope. The blc gene is poorly transcribed, suggesting that the normal concentration of Blc in the outer membrane is low.
Other findings indicate an implication of Blc in bacterial host pathogenesis (Bishop, 2000). The blc genes of some Enterobacteriaceae are physically linked to the ampC gene, which encodes a serine β-lactamase on the chromosome but also appears to be genetically recombined into different plasmids. This co-localisation suggests that the blc gene may be involved in antibiotic resistance. Furthermore, bacterial lipocalins play a role in the host immune response as many components of the bacterial cell envelope provide so-called pathogen-associated molecular patterns for surveillance. One relevant component is the N-acyl-S-sn-1,2-diacylglycerylcysteine modification at the N-terminus of the bacterial lipoproteins, permitting macrophages and other immune cells to recognize Blc via CD14 and the Toll-like receptor 2.
The first crystal structure of an N-terminally extended version of a Blc, so-called Blc-X (Campanacci et al. 2004), revealed the β-barrel fold characteristic for the lipocalin family and was followed by a second crystal structure of Blc-X in complex with the fatty acid vaccenic acid (Campanacci et al., 2006). Both structures belong to the space group P212121 with isomorphous unit cell parameters and an overall r.m.s. deviation of 0.1 Å for 167 Cα atoms.
The crystal structures of Campanacci et al. indicated that Blc was a dimeric protein. This prediction was based on the identification of a tight pairwise contact of several side chains and a buried surface of 786 Å2 and 825 Å2, respectively, of the two distinct Blc-X molecules A and B within the asymmetric unit, and also on static light scattering measurements in solution. Notably, the fatty acid ligand was bound in the cavity of just one molecule of the dimer and involved in only a few additional contacts to the other molecule, which was explained by the asymmetric interaction of the two Blc-X monomers. Yet, binding of vaccenic acid did not lead to detectable conformational changes within the Blc-X dimer (Campanacci et al., 2006).
The present inventors, however, studied the biochemistry and structure of a recombinant Blc without N-terminal extension overproduced with a different E. coli expression vector, and found striking evidence that Blc behaves as a stable monomer in solution. The previously described dimerization is, therefore, likely the result of a cloning artifact. This surprising finding is one of the bases of the present invention.